N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 - 17 Jan 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

EC Number:

263-218-7

EC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Cas Number:

61792-31-2

Molecular formula:

C17H36N2O2

IUPAC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Specific details on test material used for the study:

Freeze-dried test material was used to ensure that the purity was sufficiently high.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM reconstituted three-dimensional human epidermis (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

freeze-dried test material

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Ashland, MA, USA)
- Tissue batch number: 32138
- Date of initiation of testing: 13 Jan 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature and 60 min at 37.0 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL, 1 mL/well
- Incubation time: Approx. 3 h at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.721 ± 0.052 (acceptance criteria < 1.0 - 3.0)
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 7.67 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show reducing capacity 60 min after MTT incubation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Single experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test item is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The skin was moistened with 25 µL Milli-Q-water and 27.1 - 35.5 mg of the solid test item were added on top of the skin tissues.

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Duplicates for each treatment and control group (3 and 60 min)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control was 1.677 ± 0.103 for the 3 min exposure and 1.799 ± 0.001 for the 1 h exposure and fell within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and within the laboratory historical control data range (1.258 – 2.414 for 3 min exposure and 1.317 – 2.371 for 1 h exposure).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 15% compared to the negative control (9.2% for 3 min exposure and 12.0% for 1 hour exposure).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates for the negative control was ≤ 30% (values between 0.1 - 8.3). The CV between replicates of the test item tissues was > 30% for the 1 hour exposure. Since all individual viabilities were in the same sub-category, the test outcome was considered to be valid.

Any other information on results incl. tables

Table 1: Mean absorption data

	3-minute application			1-hour application
	A (OD570)	B (OD570)	Mean ± SD	A (OD570)	B (OD570)	Mean ± SD
Negative control	1.75	1.604	1.677 ± 0.103	1.8	1.798	1.799 ± 0.001
Test item	1.583	1.635	1.609 ± 0.037	1.646	1.053	1.349 ± 0.419
Positive control	0.248	0.158	0.203 ± 0.063	0.147	0.186	0.166 ± 0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

Table 2: Mean tissue viability data

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	96	75
Positive control	12	9.2

Table 3: Coefficient of Variation between tissue replicates

	3 minute	1 hour
Negative control	8.3	0.1
Test item	3.2	36
Positive control	36	21

CV (%) = 100 - [(lowest OD570 / highest OD570) x 100%]

Table 4: Historical control data

	Negative control		Positive control
	3-minute treatment(OD570)	1-hour treatment(OD570)	3-minute treatment(OD570)	1-hour treatment(OD570)
Range	1.258 – 2.414	1.317 – 2.371	0.083 – 0.512	0.070 – 0.298
Mean	1.71	1.73	0.18	0.14
SD	0.21	0.20	0.09	0.04
n	104	107	102	100

SD = Standard deviation

n = Number of observations

The historical control data range of the controls were obtained by collecting all data over the period of Nov 2016 to Nov 2019.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive according to Regulation (EC) No. 1272/2008.

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.